Sonication to selectively lyse different cell types

ABSTRACT

A sonication apparatus is directed to a microfluidic-based system to automate differential extraction of specific cell types within a mixed sample. The microfluidic-based system includes a sonication module for selective cell lysis, separating means to eliminate centrifugation, high surface area pillar chip modules to purify DNA from a cell lysate, and microfluidic circuitry to integrate the steps in an automated platform.

RELATED APPLICATIONS

This application claims priority of U.S. provisional application, Ser.No. 60/504,072, filed Sep. 19, 2003, and entitled “MicrofluidicDifferential Extraction Instrument”, by this same inventor. Thisapplication incorporates U.S. provisional application, Ser. No.60/504,072 in its entirety by reference.

FIELD OF THE INVENTION

The invention relates to a method and apparatus for lysing cells usingsonication. In particular, the invention relates to selectively lysingdifferent cell types using sonication.

BACKGROUND OF THE INVENTION

Cell lysis is the destruction, or disruption, of a cell's membrane orwall, which breaks open the cell and exposes its contents. Manytechniques are available for the disruption of cells, including physicaland detergent-based methods. Physical lysis often requires expensive,cumbersome equipment and involves protocols that are difficult to repeatdue to variability in the apparatus. Detergent-based methods are ofteneasier-to-use with more efficient protocols than physical methods.

Sonication is one method of physically lysing cells. Sonication usespulsed, high frequency sound waves to agitate and lyse cells, bacteria,spores, and finely diced tissue. The sound waves are delivered using anapparatus with a vibrating probe that is immersed in the liquid cellsuspension. Mechanical energy from the probe initiates the formation ofmicroscopic vapor bubbles that form momentarily and implode, causingshock waves to radiate through the sample. The sonic energy delivered toa sample using this method is variable, and not repeatable for anecessary level of precision.

Cells can be treated with various agents to aid in the disruptionprocess. For example, lysis can be promoted by suspending cells in ahypotonic buffer, which cause them to swell and burst more readily underphysical shearing. Alternatively, processing can be expedited bytreating cells with glass beads in order to facilitate the crushing ofthe cell walls.

The resistance of cells and viruses to lysis or disruption is based onthe characteristics of the cell membrane, cell wall, or coat protein.The various chemical, enzymatic, and mechanical or physical approacheshave been utilized to non-specifically lyse cells and viruses. However,in some applications, it is desirable to lyse one specific cell type orvirus in a mixed sample of two or more cells and viruses. One suchapplication is DNA typing.

DNA typing has been an invaluable tool for forensic science.Applications including linking a suspect to a crime site or a victim,identifying a perpetrator via a “cold hit” in a networked crimelaboratory DNS database, identifying a victim or human remains, andproving the innocence of wrongly incarcerated prisoners by analyzingarchived evidence. Sample types and matrices can vary considerably, andthe entire sample preparation process can be very time consuming andlabor intensive.

Rape kits, containing swab samples of biological evidence collected inhospitals from victims of sexual assault, are amongst the most common,yet difficult, sample types to process since the swabs potentiallycontain a mixture of female epithelial cells and male sperm cells.Differential extraction is applied to separate the two distinct celltypes into male and female cell lysate fractions, and extract and purifythe DNA from each fraction. A resultant genetic profile of the male DNAis compared to that of a suspect, if available, or screened through thecrime laboratory DNA database.

The conventional method for separating epithelial cells from sperm cellsinvolves selective lysis using a combination of enzymes, chemicals,heat, and centrifugation. In a mixed sample, the epithelial cells arelysed first due to their lack of a protective coat, the sperm cells arepelleted using centrifugation, the epithelial cell lysate is removed,the sperm cells are re-suspended, and the sperm cells are lysed usingmore stringent enzymatic, chemical, and heat conditions. Thisconventional process takes hours, sometimes days. New cost-effective andefficient methods and instrumentation need to be developed and validatedfor practical low-cost, low processing time, high-throughput solutions.

SUMMARY OF THE INVENTION

In one aspect of the present invention, a method of selectively lysing aspecific type of cell includes providing a sample including at least twodifferent cell types, wherein a first cell type is lysed at a firstsonication energy, applying the first sonication energy to the samplethereby lysing the first cell type to form a first lysate, separatingthe first lysate from the sample, and lysing a second cell type to forma second lysate. The second cell type can be lysed at a secondsonication energy and lysing the second cell type comprises applying thesecond sonication energy to the sample. Lysing the second cell type canalso include adding an additive to the sample prior to applying thesecond sonication energy. The first sonication energy can be lower thanthe second sonication energy. Lysing the second cell type can compriseapplying a chemical treatment to the second cell type. The method canalso include adding an additive to the sample prior to applying thefirst sonication energy. The first cell type can comprise an epithelialcell. The first cell type can comprise a virus, a microbe, or a spore.The second cell type can comprise a sperm cell. The second cell type cancomprise a virus, a microbe, or a spore. The method can also includeperforming protein purification on the first lysate. The method can alsoinclude performing protein purification on the second lysate. The secondcell type can remain intact after application of the first sonicationenergy. The sample can include N different cell types, each cell type islysed at a different sonication energy. The method can also includeapplying a sonication energy corresponding to a particular cell type andseparating the resulting lysate, and repeating the steps to generate N-1lysates. Each successive application of sonication energy to the samplecan utilize a sonication energy that is higher than the previousapplication. The method can also include adding an additive to thesample prior to one or more of the N-1 applications of the correspondingsonication energy. Applying the first sonication energy, separating thefirst lysate, and lysing the second cell type can be automated. Themethod can also include automating a protein purification of the firstlysate. The method can also include automating a protein purification ofthe second lysate. Separating the first lysate from the sample cancomprise using a filter that passes the first lysate and blocks thesecond cell type. Separating the first lysate from the sample cancomprise performing centrifugation on the sample to form a sperm cellpellet and removing the first lysate. Providing a sample can compriseplacing a sample matrix and applying a third sonication energy to thesample matrix to release the sample from the sample matrix.

In another aspect of the present invention, a method of selectivelylysing a specific type of cell includes providing a sample including atleast two different cell types, wherein a first cell type is lysed at afirst sonication energy, automatically applying the first sonicationenergy to the sample thereby lysing the first cell type to form a firstlysate, automatically separating the first lysate from the sample, andautomatically lysing a second cell type to form a second lysate. Themethod can also include automating a protein purification of the firstlysate. The method can also include automating a protein purification ofthe second lysate. Providing the sample can be automated. The secondcell type can be lysed at a second sonication energy and automaticallylysing the second cell type comprises automatically applying the secondsonication energy to the sample. Automatically lysing the second celltype can also include automatically adding an additive to the sampleprior to automatically applying the second sonication energy.Automatically lysing the second cell type can comprise automaticallyapplying a chemical treatment to the second cell type. The method canalso include automatically adding an additive to the sample prior toautomatically applying the first sonication energy. The sample caninclude N different cell types, each cell type is automatically lysed ata different sonication energy. The method can also include automaticallyapplying a sonication energy corresponding to a particular cell type andautomatically separating the resulting lysate, and automaticallyrepeating the steps N-1 times to generate N-1 lysates. Each successiveapplication of sonication energy to the sample can utilize a sonicationenergy that is higher than the previous application. The method can alsoinclude automatically adding an additive to the sample prior to one ormore of the N-1 applications of the corresponding sonication energy.Automatically separating the first lysate from the sample can compriseusing a filter that passes the first lysate and blocks the second celltype. Automatically separating the first lysate from the sample cancomprise automatically performing centrifugation on the sample to form asperm cell pellet and removing the first lysate. Providing a sample cancomprise placing a sample matrix and applying a third sonication energyto the sample matrix to release the sample from the sample matrix.

In yet another aspect of the present invention, an apparatus toselectively lyse a specific type of cell includes a sonication chamberto receive a sample and to provide a first sonication energy, the samplehaving at least two different cell types, wherein a first cell type islysed at the first sonication energy to form a first lysate, aseparating means to separate the first lysate from the sample, acollection vessel to collect the first lysate, and microfluidiccircuitry to couple the sonication chamber, the separating means, andthe collection vessel. The first cell type can comprise an epithelialcell. The first cell type can comprise a virus, a microbe, or a spore.The separating means can comprise a filter that passes the first lysateand block the remaining sample. The remaining sample can include asecond cell type, the second cell type is lysed at a second sonicationenergy. Microfluidic circuitry can provide the remaining sample with thesecond cell type to the sonication chamber to be lysed. The sonicationchamber can provide the second sonication energy to lyse the second celltype, thereby forming a second lysate. The apparatus can also include apurification chip coupled to the sonication chamber to receive thesecond lysate and collect a DNA from the second lysate. The apparatuscan be automated. The second cell type can comprise a sperm cell. Theapparatus can also include a second sonication chamber such that themicrofluidic circuitry provides the remaining sample with the secondcell type to the second sonication chamber to be lysed. The secondsonication chamber can provide the second sonication energy to lyse thesecond cell type, thereby forming a second lysate. The apparatus canalso include a purification chip coupled to the second sonicationchamber to receive the second lysate and collect a DNA from the secondlysate. The apparatus of can also include a purification chip to receivethe lysate and collect a DNA from the lysate. The sample can include Ndifferent cell types, each cell type is lysed at a different sonicationenergy. The apparatus can also include N-1 or less additional sonicationchambers to apply N-1 or less different sonication energies to thecorresponding N-1 or less different cell types. The apparatus can alsoinclude N or less purification chips to receive N or less lysates fromthe N or less sonication chambers. The separating means can comprisemeans for performing centrifugation on the sample to form a sperm cellpellet. The sonication chamber can provide a third sonication energy toa sample matrix, thereby releasing the sample from the sample matrix.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a conceptual block diagram of a sonication apparatusto selectively lyse a mixed sample using sonication according to thepreferred embodiment of the present invention.

FIG. 2 illustrates a conceptual block diagram of a sonication apparatusto selectively lyse a mixed sample using sonication according to a firstalternative configuration.

FIG. 3 illustrates a conceptual block diagram of a sonication apparatusto selectively lyse a mixed sample using sonication according to asecond alternative configuration.

FIG. 4 illustrates a general method of performing selective lysis andprotein purification.

FIG. 5 illustrates an exemplary method in which a sonication apparatusof the present invention is applied to a rape kit.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

Embodiments of a sonication apparatus of the present invention aredirected to a microfluidic-based system to automate differentialextraction of specific cell types within a mixed sample. Themicrofluidic-based system includes a sonication module for selectivecell lysis, separating means to eliminate centrifugation, high surfacearea pillar chip modules to purify DNA from a cell lysate, andmicrofluidic circuitry to integrate the steps in an automated platform.

The sonication module can include one or more sonication chambers. Inthe preferred embodiment, the sonication module includes two sonicationchambers. Alternatively, one sonication chamber can be used. Stillalternatively, three or more sonication chambers can be used when amixed sample includes three or more different cell types. Each of thesesonication module configurations is discussed in greater detail below.

Conventional sonication is an established physical method for rapid andnon-specific lysing of cells and viruses. As applied in the presentinvention, sonication is used to selectively lyse cells and viruses.Sonication amplitude, duration, and frequency can be adjusted to lysecertain cell types or viruses without lysing other cell types or virusesin a mixed sample. In addition, sonication can be used in combinationwith chemical and enzymatic approaches to further achieve higher cell orvirus lysis specificity. Other additives such as glass beads can also becombined with the sonication and/or chemical and enzymatic approaches.

In one exemplary application, forensic rape kit samples typicallyinclude sperm cells and female epithelial cells. In forensicapplications, embodiments of the present invention provide an automated,microfluidic system for differential extraction of samples having amixture of female epithelial cells and male sperm cells. The sonicationapparatus of the present invention preferably includes a sonicationmodule for selective cell lysis and a silicon chip module for rapid DNAextraction, purification, and concentration. This sonication apparatusis a turn-key system in which an operator loads samples, starts theapparatus, and allows the sonication apparatus to run unattended whileperforming the entire differential extraction method. In an alternativeembodiment, the sample loading process is also automated.

In conventional methods, a mild 2 hour enzymatic/chemical step is firstused to lyse the softer epithelial cells. The sperm cells are pelletedby centrifugation and the epithelial cell lysate is removed. The spermcells are re-suspended and subjected to a strong 2 hourenzymatic/chemical step to lyse the tougher sperm cells. In the presentinvention, a few minutes of low amplitude sonication replaces the mild 2hour enzymatic/chemical step and a few minutes of high amplitudesonication replaces the strong enzymatic/chemical step. Alternativeseparating means can also be used in place of the centrifugation toseparate the epithelial cell lysate from the intact sperm cells.

The present invention provides many advantages over conventional lysingmethods. The method of the present invention is much faster thanchemical and enzymatic approaches, the method is more repeatable, themethod provides improved cell or virus lysis selectively, and the methodis amenable to automation using robotics and/or microfluidics. In thepreferred embodiment, sonication chambers and separating means areembedded in a microfluidic system.

In the preferred embodiment, each sonication chamber is of the typedescribed in U.S. Pat. No. 6,100,084. The preferred sonication chamberincludes a container with a cavity therein for retaining a sample in anultrasonic transmission medium. The cavity is closed using a membrane.An electrode and a piezoelectric material are attached to the membrane,and a voltage source is electrically connected to the electrode and thepiezoelectric material. An amount of sonication energy necessary to lysea specific cell type is a function of many factors including, but notlimited to, the amount of voltage applied to the membrane, the thicknessof the membrane, the dimensions of the container and the cavity, thematerial of the container, the material of the membrane, a compositionof the transmission medium within the cavity, a duration of the appliedvoltage, and the type of cell to be lysed. As such, the sonicationenergy required to lyse a specific cell type is experimentallydetermined and in general is a relative function of the equipment used.As used herein, the term “sonication energy” is used to define an energyamplitude, frequency, duration, or any combination thereof. Use of thepreferred sonication chamber provides te ability to regulate the appliedsonic energy with high precision.

The microfluidic system includes microfluidic circuitry to process smallliquid volumes for complex reagent metering, mixing, and biochemicalanalysis. The microfluidic system provides a closed-loop environmentwhich minimizes environmental contamination and the potential ofcompromising the integrity of the sample.

FIG. 1 illustrates a conceptual block diagram of a sonication apparatus2 to selectively lyse a mixed sample using sonication according to thepreferred embodiment of the present invention. The mixed samplepreferably includes at least two different cell types. For example, thesample can be a rape kit sample that includes female epithelial cellsand male sperm cells. A first cell type within the mixed sample is lysedat a first sonication energy, and a second cell type is lysed at asecond sonication energy. The apparatus 2 includes a first sonicationchamber 10, a second sonication chamber 20, a separating means 30, afirst purification chip 40, a second purification chip 50, a firstoutput vessel 60, and a second output vessel 70, each of which ispreferably coupled using microfluidic circuitry, which is discussed ingreater detail below. The mixed sample is placed in the first sonicationchamber 10. The first sonication chamber 10 provides the firstsonication energy to the mixed sample, thereby lysing the first celltype. The first sonication energy used to lyse the first cell type ispreferably lower than the second sonication energy necessary to lyse thesecond cell type. As a result, after the first sonication energy isapplied to the sample, the sample includes the first cell type lysate,referred to as a first lysate, and the second cell type which is stillintact. The separating means 30 separates the first lysate from themixed sample. The first lysate passes through the separating means 30and is directed to the first purification chip 40. The separating means30 preferably includes pores or openings that each have a diameter thatis less than the diameter of the intact second cell type. Separation ofa lysate from a sample is performed using any conventional separatingmeans. Examples of such separating means include, but are not limitedto, a filter, a membrane, magnetic beads, a frit, or any other means forseparating a lysate from an intact cell type. The intact second celltype is blocked by the separating means 30 and is preferably directed tothe second sonication chamber 20 by back flowing a buffer through theseparating means 30.

The first purification chip 40 purifies and concentrates DNA from withinthe first lysate such that a first DNA is collected and the remainingportion of the first lysate passes through as waste. The collected firstDNA is collected in the first output vessel 60.

The second sonication chamber 20 provides the second sonication energyused to lyse the second cell type. The resulting second lysate ispreferably directed to the second purification chip 50. The secondpurification chip 50 purifies and concentrates DNA from within thesecond lysate such that a second DNA is collected and the remainingportion of the second lysate passes through as waste. The collectedsecond DNA is collected in the second output vessel 70. Each of thepurification chips 40 and 50 are preferably of the type described inU.S. Pat. No. 5,707,799 and U.S. Pat. No. 5,952,173, which are bothhereby incorporated by reference.

Although the preferred sonication apparatus 2 is illustrated anddescribed in terms of selectively lysing two different cell types, it isunderstood that the mixed sample can include more than two differentcell types and that the sonication apparatus can selectively lyse morethan two cell types. In this case, the number of sonication chambersused is determined by the number of anticipated different cell types tobe lysed. Each cell type to be lysed must be lysed at a differentsonication energy than the other cell types within the mixed sample. Iftwo cell types are lysed at the same sonication energy, then it is notpossible to lyse one without lysing the other using this sonicationmethod. However, cell types that are uniquely lysed at a givensonication energy, can be selectively lysed using this sonicationmethod.

In an alternative embodiment, lysing of particular cell types can befacilitated by adding additives to the sample prior to any of thesonication steps. As with empirically determining the specificsonication energy required to lyse a particular cell type, the propertype and amount of additives to be used is also determinedexperimentally. Examples of additives include glass beads, chemicals,enzymes, or the addition of heat.

In still another alternative embodiment, the last remaining cell type inthe original mixed sample can be lysed using conventional chemicaland/or enzymatic lysing methods. For example, where a mixed sampleincludes two different cell types to be lysed, the first cell type islysed using the aforementioned sonication method at the first sonicationenergy, and the second cell type is lysed using a chemical/enzymaticlysing method. As another example, where a mixed sample includes threedifferent cell types, two of the cell types can be selectively lysed asdescribed above in relation to the sonication apparatus 2. The thirdcell type can be lysed using any conventional lysing method, includingsonication, chemical, or enzymatic.

Multiple sonication apparatuses 2 can also be coupled together inparallel. In such a configuration, multiple mixed samples can beprocessed in parallel to increase throughput.

It is also understood that various alternative combinations ofsonication chambers, separating means, and purification chips can beused to selectively lyse different cell types and to collect DNA fromthe resulting lysates. FIGS. 2 and 3 illustrate two such alternativeconfigurations.

FIG. 2 illustrates a conceptual block diagram of a sonication apparatus100 to selectively lyse a mixed sample using sonication according to afirst alternative configuration. The alternative sonication apparatus100 operates similarly to the preferred sonication apparatus 2 exceptthat the two sonication chambers 10 and 20 are replaced by a singlesonication chamber 110. In operation, the first sonication chamber 110receives the mixed sample and the first sonication energy is applied,thereby lysing the first cell type and producing the first lysate. Thefirst lysate is separated from the mixed sample by the separating means30, such that the first lysate passes through the separating means 30and the intact second cell type is blocked by the separating means 30.The first lysate is directed to the first purification chip 40 asdescribed above in relation to the preferred sonication apparatus 2. Theblocked second cell type is back flushed from the separating means 30 tothe sonication chamber 110. The sonication chamber 110 applies thesecond sonication energy to the second cell type, thereby lysing thesecond cell type to produce the second lysate. The second lysate isdirected to the second purification chip 50 as described above inrelation to the preferred sonication apparatus 2.

FIG. 3 illustrates a conceptual block diagram of a sonication apparatus200 to selectively lyse a mixed sample using sonication according to asecond alternative configuration. The second alternative sonicationapparatus 200 includes a single sonication chamber 210 and a singlepurification chip 240. The sonication chamber 210 receives the mixedsample and provides the first sonication energy as described in detailabove. The first lysate passes through the separating means 230 to thepurification chip 240. The purification chip 240 purifies andconcentrates the first DNA from within the first lysate and theremaining portion of the first lysate passes through as waste. Thecollected first DNA is flushed from the purification chip 240 and iscollected in the first output vessel 260. Flushing the first DNA alsoacts to clean the purification chip 240. Alternatively, after the firstDNA is flushed from the purification chip 240 to the first output vessel260, a cleaning step is performed in which the purification chip 240 isrinsed with a cleaning solution to remove any remaining first lysate.The cleaning solution and any accompanying waste is removed from thesonication apparatus 200 using the microfluidic circuitry includedtherein.

The separating means 230 blocks the intact second cell type and theintact second cell type is collected in the sonication chamber 210. Thesecond sonication energy is provided by the sonication chamber 210,thereby lysing the second cell type. The second lysate passes throughthe separating means 230 into the purification chip 240. The second DNAis purified and collected within the purification chip 240 while theremaining portion of the second lysate passes through as waste. Thecollected second DNA is collected in a second output vessel 270.

It is understood that multiple first alternative sonication apparatuses100 can be coupled together in parallel, and that multiple secondalternative sonication apparatuses 200 can be coupled together inparallel. Further, one or more sonication apparatuses 2, one or moresonication apparatus 100, and/or one or more sonication apparatuses 200can be coupled together in parallel to simultaneously process multiplemixed samples.

Each sonication apparatus 2, 100, and 200 are preferably configured asmicrofluidic cassettes. A microfluidic cassette avoids the necessity ofcomplicated, expensive robotic devices, while improving theeffectiveness and efficiency of fluid metering, mixing, and dispensing.The cassette can include a multilayer microfluidic plastic block withfluidic circuitry and multiple independent active valves, syringedrives, a sonication lysis module including one or more sonicationchambers as described above, one or more DNA purification chips, reagentreservoirs, electronic hardware, and custom user interface. Fluid flowthrough the microfluidic cassette can be pressure-driven andcomputer-controlled. As such, each sonication apparatus can becompletely automated.

The microfluidic cassette can include a liquid-handling module and apneumatic module. Each of these modules can be comprised of severallayers of machined polycarbonate plastic parts, which are sandwiched andsealed together using laser cut sheets of silicone gasket material. Thesilicone gasket material serves to seal the layers in each module andalso to function as diaphragm valves in the liquid-handling module. Tensto hundreds of independently controlled valves, working systematicallyto direct the flow of sample and reagents, can be created.

Syringe pumps can be used as a drive mechanism for moving, mixing,aspirating, and dispensing boluses of liquid between locations in theliquid-handling block. The syringe driver boards control a stepper motorthat moves the syringe plungers. Since the full stroke of the syringeshas 48,000 steps, high precision fluid metering can be accomplished. Forexample, the resolution for displacement of fluid using a 1 ml syringebarrel is 0.0208 ul per step. A variety of syringe sizes can beincorporated to accommodate fast, large volume movement and precisesmall volume metering.

Peristaltic pumps can also be used as the drive mechanism. Theperistaltic pump can achieve continuous flow and minimizes problems ofair in the lines.

The liquid-handling module can be mounted on the pneumatic module witho-rings around each pressure outlet to seal the pneumatic path. Valvesin the liquid-handling module are opened and closed by electricalactuation of three-way solenoid valves mounted on the backside of thepneumatic module. The liquid-handling module preferably include inletand outlet ports, microchannels, mixing chambers, at least onesonication chamber, and at least one DNA purification chip. Solutionsand reagents can be included within the cassette, or they can beintroduced from lines connected to external bulk containers.

A simple and intuitive Graphical User Interface (GUI) offers control tothe system hardware components. The GUI enables a user to fully controlthe syringe drive motion and speed, and all the pneumatically actuatedmembrane valves. The GUI also enables the user to script, store, load,and run protocols, which can be edited and modified throughout thecourse of protocol optimization.

The microfluidic cassette can be configured to process multiple samplessimultaneously. In one embodiment, the cassette can process 10 samplessimultaneously per run. If one run takes 30-45 minutes, then up to 480samples can be processed in a 24 hour period. The automation process canbe extended to use an automated feeder of samples to the microfluidiccassette.

The microfluidic cassette can also include a DNA purification module.The DNA purification module preferably uses a DNA purification chip. Thepurification chip is preferably embedded in the plastic microfluidiccassette. The purification chip is preferably a micromachined siliconstructure having micropillars. The micropillars create a high surfacearea within a collection chamber. Nucleic acids are captured on thepillars, washed, and released in a small elution volume using standardchaotropic salt chemistry. Advantages to this flow through processinclude amenability to a system integration in a microfluidic platformand rapid extraction of nucleic acids from large volume samples. Inaddition, the high concentration effect permits using less sample andconsequently less PCR (Polymerase Chain Reaction) reaction mix for PCRamplification.

Another advantage of the micropillar chip for DNA purification andconcentration is the ability to produce compact arrays of these chipsfor high throughput purification. An array of purification chips can beharbored in a microfluidic circuit. Each chip can have a dedicated lineand essentially be isolated form one another. Such a configuration canbe accomplished using microfluidics in which an intricate network ofchannels is confined to a relatively small area. Such an array can havebroad applications for preparing DNA from a wide variety of sampletypes. An exemplary array of DNA purification chips includes an 8×12array of 96 micropillar chips.

FIG. 4 illustrates a general method of performing selective lysis andprotein purification. At a step 400, a mixed sample is provided to asonication chamber. The mixed sample preferably has two or moredifferent cell types, each cell type is lysed at a different sonicationenergy. For example, a first cell type lyses at an associated firstsonication energy, a second cell type lyses at an associated secondsonication energy, and so on. At a step 410, a buffer solution is addedto the mixed sample within the sonication chamber. At a step 420, afirst sonication energy is applied to the mixed sample, thereby lysing afirst cell type within the mixed sample. After sonication, the mixedsample comprises a first lysate, which is the lysed first cell type, anda remaining portion of the mixed sample. The remaining portion of themixed sample includes intact cells for each cell type that has asonication energy greater than the first sonication energy alreadyapplied.

At a step 430, the first lysate is separated from the mixed sample.Preferably, the separating means allows the first lysate to pass whileblocking the remaining portion of the mixed sample. At a step 440, thefirst lysate is passed through a purification chip to purify andconcentrate a first protein included within the first lysate. The firstprotein is collected within the purification chip while a remainingportion of the first lysate passes through the purification chip aswaste. The first protein collected in the purification chip ispreferably flushed and collected as a first purified protein sample.

Additional cell types in the remaining mixed sample can also be lysed.If a single remaining cell type is in the remaining mixed sample, thenthe remaining cell type can be lysed either using sonication or using aconventional lysing method such as a chemical and heat combination. Ifmore than one different cell type remain in the remaining samplemixture, than the steps 410 through 440 are repeated for each remainingcell type starting with the cell type with the lowest lysing sonicationenergy and working upward. Again, the last remaining cell type can belysed using sonication or other conventional lysing method.

At a step 450, a last remaining cell type within the remaining mixedsample is prepared for sonication. Preferably, the last remaining celltype is lysed using sonication. In this preferred case, a buffersolution is added to the remaining mixed sample in preparation forsonication. Alternatively, the last remaining cell type is lysed using aconventional lysing method. In this alternative case, chemical additivesare added to the remaining mixed sample. At a step 460, the remainingcell type is lysed to form a final lysate. At a step 470, the finallysate is passed through a purification chip to purify and concentrate afinal protein included within the final lysate. The final protein iscollected within the purification chip while a remaining portion of thefinal lysate passes through the purification chip as waste. The finalprotein collected in the purification chip is preferably flushed andcollected as a final purified protein sample.

FIG. 5 illustrates an exemplary method in which a sonication apparatusof the present invention is applied to a rape kit. In such anapplication, the rape kit includes a mixed forensic rape sample havingfemale epithelial cells and male sperm cells on a matrix. The matrix caninclude a swab or a swatch. It is desired to isolate the male DNA fromthe sperm cells, the female DNA from the epithelial cells, or both. Inthe preferred embodiment, two sonication steps are included. A firstsonication step uses a mild treatment to lyse epithelial cells but notsperm cells. A second sonication step uses a harsh treatment to lysesperm cells. In an alternative embodiment, a chemical/heat treatmentstep is implemented to lyse the sperm cells, thereby eliminating therequirement for a second sonication chamber on the integrated platform.

In a step 500, the swab or swatch with the mixed rape sample is placedin the sonication chamber. At a step 510, a buffer solution is added tothe mixed rape sample. Preferably, the buffer solution is a 0.5-1 ml 10mM Tris-HCL with a pH of about 8.0. Alternatively, the buffer solutionis water. Still alternatively, the sonication chamber also includesglass beads. It is understood that the steps 500 and 510 can be reversedsuch that the sonication chamber already includes the buffer solutionprior to adding the mixed rape sample. At a step 520, a first sonicationenergy is applied to the mixed rape sample. As described above, thespecific amount of sonication energy necessary to lyse the epithelialcells is dependent on the specifications of the particular sonicationchamber used. Application of the first sonication energy releases boththe epithelial cells and the sperm cells from the matrix into the buffersolution. The first sonication energy is sufficient to selectively lysethe epithelial cells, however the more durable sperm cells remainintact. Sonication at the first sonication energy is considered a mildsonication. After application of the first sonication energy, the buffersolution includes the lysed epithelial cells and intact sperm cells.

At a step 530, the swab or swatch is preferably removed and the lysedepithelial cells are separated from the intact sperm cells. Theseparating means preferably includes a filter with pores or openingseach with a diameter of about 2 um. The sperm cells, which have about a5 um diameter, are blocked by the separating means, while the lysedepithelial cells pass through the separating means to a femalecollection vessel. In general, the diameter of the filter pores oropenings is large enough to allow the epithelial cell lysate to pass,but small enough to prevent the intact sperm cells from passing. Thesonication chamber is preferably rinsed with buffer to move theepithelial cell lysate through the separating means. At a step 540, thelysed epithelial cells are preferably mixed with an equal volume of bindsolution and passed through a purification chip where the femaleepithelial DNA is purified and concentrated from the remainingepithelial cell lysate. The remaining epithelial cell lysate passesthrough the purification chip as waste. The epithelial cell DNA iscollected from the purification chip.

At a step 550, the intact sperm cells are back flushed from theseparating means to a second sonication chamber and a 0.2 molar NaOH, orbasic solution, is added. Alternatively, the intact sperm cells are backflushed from the separating means to the sonication chamber where theepithelial cells were previously lysed. The NaOH solution preferablyincludes 50 mM DTT. Alternatively, proteinase K can also be added to thepreferred basic solution. At a step 560, a second sonication energy isapplied to lyse the sperm cells. The second sonication energy is greaterthan the first sonication energy used to lyse the epithelial cells.Sonication at the second sonication energy is considered a harshsonication. At a step 570, the lysed sperm cells are passed through theseparating means and directed a second purification chip. Alternatively,the lysed sperm cells can first be collected in a male collectionvessel, and later directed to the second purification chip. The lysedsperm cells are preferably mixed with an equal volume of bind solutionand passed through the second purification chip where the male sperm DNAis purified and concentrated from the remaining sperm cell lysate. Theremaining sperm cell lysate passes through the purification chip aswaste. The sperm DNA is collected from the purification chip.Alternatively, a single purification chip is used to collect both theepithelial DNA and the sperm DNA. In some applications, DNA purificationis not required and the collected intact sperm can be removed from thesonication apparatus prior to application of the second sonicationenergy.

In an alternative embodiment, the male sperm cells can be lysed bymethods other than sonication. For example, instead of performing thesteps 550 and 560, the intact sperm cells are collected in a malecollection vessel, basic solution is added, and the male collectionvessel is heated to a predetermined temperature for a predeterminedduration to lyse the male sperm cells.

In another alternative embodiment, after the first sonication, theintact sperm cells are separated from the epithelial cell lysate bysubjecting the mixture to centrifugation. The epithelial cell lysate isremoved from the sperm cell pellet. The sperm cell pellet isre-suspended and subjected to sonication at the second sonication energyto lyse the sperm cells. It should be understood that alternativeseparating means can be used, as described above.

As previously discussed, a systematic approach is taken to develop themost appropriate protocol for the differential extraction method. First,sonication threshold levels for sperm cells and epithelial cells aredetermined. It is known that sperm cells are more resistant tosonication than epithelial cells. DTT is known to break down thedisulfide bonds in the sperm coat. Sonication with the addition of DTTcan be used to facilitate sperm cell lysis. A typical procedure forspore lysis uses glass beads during sonication, and although the useglass beads during sonication does lyse sperm cells, glass beads arepreferably avoided for sperm cell lysis. Selective lysis of theepithelial cells is preferably accomplished using mild sonicationwithout DTT.

It should be understood that the lysates can be purified using meansother than the described purification chips. For example, the epithelialand sperm cell lysates can be subjected to DNA purification using thepurification chip, glass membrane, glass column, organic extraction,ethanol precipitation, or any other method known to purify nucleicacids.

The present invention has been described in terms of specificembodiments incorporating details to facilitate the understanding of theprinciples of construction and operation of the invention. Suchreference herein to specific embodiments and details thereof is notintended to limit the scope of the claims appended hereto. It will beapparent to those skilled in the art that modifications may be made inthe embodiment chosen for illustration without departing from the spiritand scope of the invention.

1. A method of selectively lysing a specific type of cell, the methodcompnsing: a. providing a sample to a self-contained, automated system,the sample includes at least two different cell types, wherein a firstcell type is lysed at a first sonication energy; b. automaticallyapplying the first sonication energy to the sample by lysing meanswithin the automated system thereby lysing the first cell type to form afirst lysate and leaving intact one or more non-lysed cell types; c.automatically separating the first lysate from the sample including theintact one or more non-lysed cell types by separating means within theautomated system, thereby forming a separated sample including theintact one or more non-lysed cell types; d. automatically moving theseparated sample to the lysing means by a back-flow capability andmicrofluidic circuitry within the automated system, wherein themicrofluidic circuitry includes the back-flow capability for moving theseparated sample from the separating means to the lysing means; and e.automatically lysing a second cell type from the separated sample bylysing means within the automated system thereby lysing the second celltype to form a second lysate.
 2. The method of claim 1 furthercomprising automating a protein purification of the first lysate.
 3. Themethod of claim 1 further comprising automating a protein purificationof the second lysate.
 4. The method of claim 1 wherein providing thesample is automated.
 5. The method of claim 1 wherein the second celltype is lysed at a second sonication energy and automatically lysing thesecond cell type comprises automatically applying the second sonicationenergy to the sample.
 6. The method of claim 5 wherein automaticallylysing the second cell type further comprises automatically adding anadditive to the sample prior to automatically applying the secondsonication energy.
 7. The method of claim 1 wherein automatically lysingthe second cell type comprises automatically applying a chemicaltreatment to the second cell type.
 8. The method of claim 1 furthercomprising automatically adding an additive to the sample prior toautomatically applying the first sonication energy.
 9. The method ofclaim 1 wherein the sample includes N additional different cell types,each additional cell type is automatically lysed at a differentsonication energy, wherein N is an integer greater than or equal to one.10. The method of claim 9 further comprising automatically applying aparticular sonication energy to lyse a particular cell type andautomatically separating the resulting lysate from the sample for each Nadditional different cell types, thereby forming N additional lysates.11. The method of claim 9 wherein each successive application ofsonication energy to the sample utilizes a sonication energy that ishigher than the previous application.
 12. The method of claim 9 furthercomprising automatically adding an additive to the sample prior to oneor more applications of the sonication energy.
 13. The method of claim 1wherein automatically separating the first lysate from the samplecomprises using a filter that passes the first lysate and blocks thesecond cell type.
 14. The method of claim 1 wherein automaticallyseparating the first lysate from the sample comprises automaticallyperforming centrifugation on the sample to form a second cell typepellet and removing the first lysate.
 15. The method of claim 1 whereinproviding a sample comprises providing a sample matrix and applying thefirst sonication energy to the sample matrix to release the sample fromthe sample matrix.